In the present application, the amino acid residues/positions in an immunoglobulin heavy-chain variable domain will be indicated with the numbering according to Kabat. For the sake of convenience, FIG. 1 gives a table listing some of the amino acid positions that will be specifically referred to herein and their numbering according to some alternative numbering systems (such as Aho and IMGT. Note: unless explicitly indicated otherwise, for the present description and claims, Kabat numbering is decisive; other numbering systems are given for reference only).
With regard to the CDRs, as is well-known in the art, there are multiple conventions to define and describe the CDRs of a VH or VHH fragment, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the location of the structural loop regions). Reference is for example made to the website http://www.bioinf.org.uk/abs/. For the purposes of the present specification and claims, even though the CDRs according to Kabat may also be mentioned, the CDRs are most preferably defined on the basis of the Abm definition (which is based on Oxford Molecular's AbM antibody modelling software), as this is considered to be an optimal compromise between the Kabat and Chothia definitions. Reference is again made to the website http://www.bioinf.org.uk/abs/.
Inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC) are chronic, disabling and progressive diseases. Most non-biological drug therapies such as aminosalicylates, steroids and immunomodulators provide symptomatic improvement but fail to stop the underlying inflammatory process and do not change the disease course. The advent of anti-tumor necrosis factor-α (anti-TNF-α) agents (infliximab, adalimumab, certolizumab pegol) has dramatically changed the way IBD is treated by changing both disease course (fewer surgeries, less hospitalizations, better quality of life, steroid sparing, greater clinical remission and mucosal healing rates in both CD and UC) and patients' quality of life and work productivity (cf. Amiot and Peyrin-Biroulet 2015 Ther Adv Gastroenterol 8:66-82). The most common route of administration for these therapeutic proteins is injection. Since most of these proteins have short serum half-lives, they need to be administered frequently or in high doses to be effective. Systemic administration is furthermore associated with an increased risk of infections. Together this results in a loss of patient compliance (cf. Singh et al. 2008 J Pharm Sci 97:2497-2523).
However, the undesirable long-term side effects and opportunistic infections, including tuberculosis and non-Hodgkins lymphoma are caused by the generalized immunosuppression and result from repeated systemic injections of the currently used monoclonal antibody treatment (Ali et al., 2013; Galloway et al., 2011; Ford & Peyrin-Biroulet, 2013; Kozuch & Hanauer, 2008; Schreiber et al., 2007; Syed et al., 2013).
Oral administration of anti-TNF-α antibodies should avoid some of these side effects.
However, these anti-TNF-α agents are complex proteins. Oral delivery results in degradation in the acidic and protease-rich environment of the gastrointestinal (GI) tract.
In using decoy proteins to protect the therapeutic antibody, a patient based study comprising polyclonal bovine colostral antibodies to human TNF alpha (AVX-470) administered via the oral route has recently been conducted by Avaxia Biologics Inc. in ulcerative colitis patients. However, the study was discontinued.
Hence, there is a need for new IBD drugs.
ISVDs (and in particular Nanobodies) that can bind to TNF and their uses are well-known in the art, for example from WO 2004/041862 and WO 2006/122786, which describe Nanobodies against TNF and their use for the prevention and/or treatment of diseases and disorders associated with and/or mediated by TNF-α or TNF-α signaling, such as inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, autoimmune hepatitis, autoimmune parotitis, diabetes type 1, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, male infertility, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, and vasculitis.
WO 2006/122786 disclosed as SEQ ID NO: 125 a specific anti-TNF-α Nanobody referred to as NC55TNF_NC7 (PMP6C11). The sequence of this prior art Nanobody is given in Table A below as SEQ ID NO: 58. Table A also gives (as SEQ ID NO: 1) the sequence of a sequence optimized version (also referred to herein as “Reference A”) of this prior art TNF binder, together with its CDRs (according to the Kabat and Abm conventions). As can be seen from the alignment in FIG. 2, this sequence optimized version contains, compared to the prior art sequence of SEQ ID NO: 58, the following mutations: Q1E, A14P, Q27F, S29F, P40A, A49S, K73N, Q75K, V78L, D82aN, K83R and Q108L (according to Kabat numbering).
WO 2015/1733256 relates to improved immunoglobulin domains, comprising C-terminal extensions preventing the binding of so-called pre-existing antibodies (“PEAs”). WO 2015/1733256 discloses SEQ ID NO: 345 as a specific anti-TNF-α Nanobody, which is referred to herein also as TNF345 (SEQ ID NO: 59). As can be seen from the alignment in FIG. 2, the sequence optimized version Reference A contains, compared to the prior art sequence of SEQ ID NO: 59, the following mutations: V11L and L89V (according to Kabat numbering).
A cocktail of toxin-neutralizing llama monoclonal VHH antibody fragments has recently been proposed for potential oral therapy (Hussack et al., 2011 J Biol Chem 286, 8961-76.). However, Dumoulin et al. (2002 Protein Sci, 11, 500-15), Harmsen et al. (2006 Appl Microbiol Biotechnol, 72, 544-51) and Hussack et al. (2012 Methods Mol Biol, 911, 417-29) alleged that the single domain llama antibody fragments appear to be greatly susceptible to proteolytic destruction within the human gastrointestinal system.
WO2007/025977 describes a treatment of chronic enterocolitis, involving in situ secretion of anti-mTNF Nanobodies by orally administered L. lactis bacteria.